BioMed Central
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Journal of Translational Medicine
Open Access
Research
Proteomic characterization of HIV-modulated membrane
receptors, kinases and signaling proteins involved in novel
angiogenic pathways
Suraiya Rasheed*, Jasper S Yan, Adil Hussain and Bruce Lai
Address: Laboratory of Viral Oncology and Proteomics Research Department of Pathology, Keck School of Medicine, University of Southern
California, 1840 N Soto St, Los Angeles, CA 90032-3626, USA
Email: Suraiya Rasheed* - ; Jasper S Yan - ; Adil Hussain - ; Bruce Lai -
* Corresponding author
Abstract
Background: Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-
infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without
HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-
tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic
processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis in vivo unless specific proteins/
enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that chronic HIV-
replication in non-endothelial cells may produce novel factors that provoke angiogenic pathways.
Methods: Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive
proteomics analyses at various stages of virus and cell growth in vitro over a period of two years. Several thousand
differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple
gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses.
Results: By functional categorization, 31 proteins were identified to be associated with various signaling events involved
in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be
essential for regeneration, neovascularization and angiogenic processes during embryonic development.
Conclusion: Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth
factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected

ers and inhibitors of angiogenesis respectively) produced
by various cell types maintain a balance between neovas-
cularization and angiogenesis programs in a cyclic man-
ner [4,5]. Exactly how abnormal angiogenic signals are
generated in vivo is not well-understood, but an imbalance
in the production of one or more critical factors can alter
the protein-protein interaction pathways and induce ang-
iogenic anomalies including inflammation, vascular
dementia, hemangioma, dysfunctional uterine bleeding,
ovarian hyperstimulation and choroidal/intraocular dis-
orders to name a few [1,6]. Angiogenesis is also critical for
cancer metastasis, diabetic blindness, age-related macular
degeneration, rheumatoid arthritis, psoriasis, and for the
development of new blood vessels that supply oxygen and
nutrients to the body when aortas are clogged (thrombo-
sis) [2,6].
In both the neoplastic and non-neoplastic diseases,
endothelial cells have been shown to express various iso-
forms of the vascular endothelial growth factors (VEGFs)
which bind to their cognate VEGF receptors (VEGFRs),
activate their associated protein tyrosine kinases (PTKs)
and stimulate endothelial cell growth through angiogenic
pathways [3,6,7]. However, endothelial cells can be acti-
vated by various cytokines, phosphorylated proteins and
other factors that are essential not only for cell growth but
also for maintaining an activated state of the stimulated
endothelial cells [2,8]. In the absence of specific cytokines
and diffusible signaling proteins, VEGF by itself is not suf-
ficient to trigger expression of numerous enzymes and
proteins required for the development of a network of

HHV8-genome itself encodes a viral G-protein-coupled
receptor (vGPCR), which activates both oncogenic and
angiogenic pathways in the presence or absence of HIV-coin-
fection [17,19,20].
Many HIV-infected patients, who may or may not be
infected with HHV8, develop intraepithelial neoplasia,
hemangiomas, lymphomas, angiosarcomas, myelodys-
plastic angiogenic syndrome and other angiopathies [21-
23]. The HIV-encoded transcriptional transactivator (Tat)
protein has been implicated in angiogenesis because it
binds VEGFR and stimulates endothelial cell growth [17].
However, its binding-affinity is not as strong as that of the
natural cellular VEGFs and the avidity of Tat interaction
with VEGFR is dependent on specific cytokines produced
locally by endothelial cells, cancer cells or other virus-
infected and uninfected cell types in vivo [10,13,24,25].
Further, the activated state of endothelial cells must be main-
tained continuously during the numerous biological proc-
esses that lead to angiogenesis. These data suggest that
while Tat synergizes the effects of many viral and cellular
factors during the complex biological processes of angio-
genesis, Tat alone or individual cytokines by themselves
do not induce angiogenesis in mice.
The molecular mechanisms involved in HIV-induced vas-
culopathies in humans are difficult, if not impossible to
study because most patients are co-infected with different
pathogenic viruses such as HSV-1, HSV11, EBV, hepatitis
B virus (HBV), hepatitis C virus (HCV), human papilloma
virus (HPV) and different bacterial and fungal microor-
ganisms. Consequently, cellular changes induced by HIV

those that are preferentially "macrophage/monocyte-
tropic" (SR personal observation). The RH9 cells do not
induce cytopathic effects but occasionally, when some
chronically infected cultures exhibit syncytia, uninfected
counterpart cells are added to maintain long-term HIV-
infected cell lines.
The choice of T-cells for HIV infection was also based on
the fact that T-cells, together with monocytes and macro-
phages present at the portal of entry in vivo are the first cell
types to be infected soon after HIV-exposure. Our experi-
ments were deliberately designed to avoid the use of pri-
mary T-cells for HIV-infection due to the genetic
heterogeneity and sample-to-sample variation in the sus-
ceptibility of freshly cultured human peripheral blood
mononuclear cells (PBMC) (SR unpublished data). Since
HIV-infected individuals harbor a variety of different
strains (present as quasispecies in vivo), we used a biolog-
ically cloned HIV strain (X4) in order to have better repro-
ducibility and consistency of results from experiment to
experiment. This methodology reduced variations in their
replication potentials.
While several HIV-infected T-cell lines or Tat-transfected
T-cell lines have been used to study HIV-infected pro-
teomes and gene expression profiles, all of these analyses
were conducted after a short time (24–48 hrs) of infection
or transfection of cells [29-32]. Given that most HIV-dis-
eases including vasculopathies are developed after several
years of chronic infection, we compared genome-wide
proteins from HIV-infected and counterpart uninfected T-
lymphocytes over a period of two years by subtractive pro-

approximately 3 months and duplicate samples from
HIV-infected and counterpart uninfected samples were
tested at 14 time points by proteomics analyses. These
samples ranged from 1.5 h to 96 days (d) post-infection
(3 h, 6 h, 12 h, 24 h, 48 h, 4 d, 10 d, 14 d, 20 d, 26 d, 28
d, 47 d and 96 d). In subsequent experiments, samples
were harvested at the peak of HIV-replication (i.e. from 10
to 26 days). Given that most HIV-associated diseases
develop after a chronic infection, we tested an additional
ten different chronically HIV-infected and uninfected
counterpart cells selected randomly over a period of two
years i.e. at various stages of virus replication and cell
growth. This large sample size was necessary in order to
select highly reproducible protein spots in multiple gels
and for testing many quality-control samples used for
standardization of experiments such as lyophilized E. coli
extract, commercially available purified proteins and a
single extract of HIV-infected and uninfected cells.
Isolation of Plasma Membrane and Extracellular Matrix
Proteins
A major goal of this study was to identify cell surface pro-
teins involved in generating HIV-modulated signals that
disrupt normal cellular functions and drive infected cells
in specific directions. Over the years our laboratory has
developed a rapid sequential extraction procedure to suc-
cessfully isolate functionally relevant and naturally occur-
ring plasma membrane and extracellular matrix proteins
[33,34]. All proteins were isolated by unbiased
approaches (i.e. without the use of special ligands, anti-
Journal of Translational Medicine 2009, 7:75 />Page 4 of 24

Coomassie Brilliant Blue for 30 minutes and de-stained in
15% (v/v) methanol, 7% (v/v) acetic acid for a minimum
of three hours. Several Coomassie-stained gels were coun-
terstained with Sypro Ruby Red (SRR) fluorescent dye
after the gels were scanned for image-analysis and double
stained gels were scanned again. Since fluorescent signals
of SRR are photostable and comparable to Cy3 and Cy5
dyes [35], this procedure enhanced the sensitivity of some
light-colored spots and reduced non-specific spot identity.
Bioinformatics and Statistical Analyses for Identification
of Angiogenic Proteins
Genome-wide protein profiles of both the infected and
uninfected counterpart cells were compared and evalu-
ated by subtractive proteomics analyses overtime i.e. at
different stages of virus and cell growth. Only those pro-
teins that were clearly identified by Matrix Assisted Laser
Desorption Ionization-Time of- Flight (MALDI-TOF)
mass spectrometry (MS) in multiple gels were included in
the final analyses. Further, any "new" proteins (i.e. hypo-
thetical proteins) identified by MS or peptide fingerprint-
ing with low Molecular Weight Search (MOWSE) Scores
(p = 0.05 or more) in any gel were excluded from the cur-
rent analyses regardless of the intensity of the stain.
All protein profiles from the HIV-infected and uninfected
cells were compared and analyzed by a variety of subtrac-
tive computer-based approaches. Integrated programs for
accuracy analyzed all proteins by calculating means and
standard deviations for quantitative evaluations of pro-
teins in both HIV-infected and uninfected controls. To
identify HIV-modulated proteins related to angiogenesis,

proteins were uploaded and function-specific pathways
were generated automatically by using IPA as well as Strat-
agene Architect programs. Although similar pathways
were constructed by the two programs, the protein-pro-
tein interaction pathways presented herein were made by
the Stratagene Architect program.
Results and discussion
Cell culture supernatants from all experimentally HIV-
infected cells showed an exponential increase in the p24
antigen levels tested over time by the enzyme-linked
immunoassays. Although many HIV-encoded proteins
(gag-p24, Tat, Rev, Vpu, Vpr, Vif, gp120, gp41 and the
polymerase) were identified by mass spectrometry (MS)
in various protein-complexes, in this study we have
focused on the identification of HIV-modulated cellular
proteins only (i.e. not encoded by viral genes).
Journal of Translational Medicine 2009, 7:75 />Page 5 of 24
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Functional Categorization of Cellular Proteins
Comprehensive MS analyses of several thousand proteins
confirmed more than 200 proteins from multiple gels run
at different phases of cell growth and virus replication
over time. Results presented herein have been consoli-
dated from proteomics data generated over a period of >
2 years. Each of the differentially regulated proteins was
functionally categorized by the use of bioinformatics pro-
grams that integrated biological information currently
located in several global databases including Ingenuity
Systems' knowledgebase of the Functional Repository of
Human Genes. We have identified 31 proteins that have

tor (VWF) (Figures 2 &3). Although numerous transcrip-
tion factors were induced de novo or upregulated post-
HIV-Infection of T-cells, in the present analysis, we have
considered the endothelial cell-specific zinc finger tran-
scription factor (ZNF71) induced by TNF alpha and
(TP53B), as important regulatory proteins that may be
necessary for the expression of cell-cycle genes/proteins
during the complex biological processes of angiogenesis
in vivo.
The VEGFR2 receptor and its growth factor ligand VEGFC
were downregulated in HIV-infected cells, although
detected only once in one of numerous acutely HIV-
infected cultures tested. The PKC-regulatory protein 143G
was expressed at a lower level in HIV infected cells com-
pared to the uninfected controls. The quantities of LAMA5
and CLR1 were not much different between the infected
and uninfected cells (Figure 3). In addition a phosphatase
(PPAC) was completely suppressed after HIV-infection
(i.e. detected only in the uninfected counterpart cells)
(Figure 4). The downregulation of PPAC is considered to
be significant because its absence is essential for maintain-
ing phosphorylation of various tyrosine kinases and acti-
vation of endothelial cell growth in vivo [36].
The biological significance of all 31 proteins identified in
this study was computed in relation to protein-interaction
networks involved in angiogenesis (p = 8 × 10
-12
). This, we
believe, is the first step toward developing a better insight
into the molecular mechanisms by which pathogenic

tion) and may be critical for the survival of the infected
cells. These proteins maintain cellular integrity during var-
ious phases of HIV replication and cell growth. Many pro-
teins that are upregulated, downregulated or induced de
novo post-HIV infection may also be necessary to compen-
sate for the loss or disruption of essential physiological
functions performed by the T-lymphocytes prior to HIV
infection.
Journal of Translational Medicine 2009, 7:75 />Page 6 of 24
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Among a diverse family of multifunctional signaling pro-
teins induced de novo in HIV-infected cells, the protein
tyrosine kinases, the serine/threonine kinases and many
regulatory enzymes appear to play major roles in T-cell
activation and global reprogramming of the transcrip-
tional and translational activities that lead to novel inter-
action pathways (Table 1).
Zeta Chain Tyrosine-Protein Kinase (ZAP-70)
The zeta chain protein tyrosine kinase (ZAP70-PTK) was
expressed exclusively in HIV-infected cells (Table 1; Figure
2). This kinase is associated with the zeta chain of the T-
cell receptor (TCR) expressed on the plasma membrane.
The tyrosine kinase activity of this receptor phosphor-
Table 1: HIV-Modulated Proteins Associated With Essential Steps During Angiogenesis
Protein names and Abbreviations Accession # P-Value related to Angiogenesis
1. Activation of T-Cells: Transcriptional and Translational Reprogramming
T-cell receptor zeta chain, tyrosine-protein kinase (ZAP-70) P43403 4 × 10
-5
TNF receptor (TNR) superfamily # 9 (TNR9) Q07011 8 × 10
-8

-12
CRK-like adapter protein (CRKL) P46109 5 × 10
-10
6. Balanced Cell Growth or Adhesion: Anti-angiogenic G-Protein Coupled Receptors
Brain-specific angiogenesis inhibitor 1 (BAI1) O14514 2 × 10
-10
Brain-specific angiogenesis inhibitor 3 (BAI3) O60242 N/A
7. Adhesion, Differentiation & Cell Migration: Focal Adhesion Kinase, Adhesion Receptor & Enzymes
Focal adhesion tryosine kinase 2 beta (FAK2) Q14289 2 × 10
-9
Alpha (V) beta (5) integrin (ITB5) P18084 2 × 10
-9
Nitric-oxide synthase (NS2A) P35228 2 × 10
-9
Fibronectin Precursor (FINC) P02751 1.5 × 10
-3
Low molecular weight phosphotyrosine protein phosphatase (PPAC) P24666 2 × 10
-9
8. Morphogenesis and Cell Migration: Laminins and other Cell Adhesion Molecules
Laminin beta-2 chain precursor (LAMB2)Upregulated P55268 2 × 10
-8
Laminin alpha-5 chain protein precursor (LAMA5) O15230 2 × 10
-10
Cadherin EGF LAG seven-pass G-type receptor 1 (CLR1/CELSR1) Q9NYQ6 N/A
Protocadherin focal adhesion targeting (FAT2) Q9NYQ8 7 × 10
-6
Golgi apparatus Protein 1 (GLG1) Q92896 N/A
9. Cell Permeability & Sprouting: Myosin Light Chain Kinase, Aggrecans & Peptidase
Myosin light chain kinase smooth muscle/non-muscle isoezymes (KMLS) Q15746 3 × 10
-12

plex-1(MHC-I) via a membrane associated lipid kinase,
phosphatidylinositol-4-phosphate3-kinase C2-beta
(PI3K) pathway (Figures 2, 5), [41,42]. Although this
interaction also affects immune evasion of HIV-infected
CD4+ T-cells, our experimentally-infected cells expressed
PI3K, concomitantly with the activation of ZAP-70 and
other protein tyrosine kinases. Co-expression of these pro-
teins is critical for efficient coupling and antigen recogni-
tion of several intracellular signal transduction molecules
and may also promote cell-to-cell contacts and increased
HIV-spread [40,43].
An interesting finding relevant to our study was that the
upregulation of ZAP-70 PTK correlates negatively with the
expression of VEGF in patients with highly malignant,
angiogenic chronic B lymphocytic leukemia (CLL) [44,45].
Although B-cell functions are not compromised by an
increase in ZAP70 kinase, its expression on the surface of
CLL cells has been linked to the increased angiogenesis
and poor prognosis of this cancer [45,46]. On the con-
trary, absence of ZAP-70 expression was a good prognosti-
cator for CLL (i.e. with less or no angiogenesis) although
VEGF was expressed [44]. These data suggest that VEGF-
independent pathways were involved in CLL malignancy.
Our proteomics and bioinformatics analyses of HIV-
infected cells are consistent with these findings since
expression of ZAP-70 PTK and other PTK-containing pro-
teins was associated with concomitant downregulation of
both the VEGF and its cognate receptor VEGFR (p = 2.6 ×
10
-3

PROT/UniProt databases. Y-axis illustrates average of nor-
malized quantity of specific protein spot computed automati-
cally by the use of PDQuest program from multiple gels.
Error bars represent one standard deviation of the range for
each protein data. Full protein names, abbreviations and
accession #s of each protein are provided in Table 1.

0
1000
2000
3000
4000
5000
6000
7000
8000
9000
ANXA6
ATS9
BAI1
BAI3
CO3
CRKL
ERB2
FAK2
FAT2
GLG1
GRB2
KMLS
KPCB

Expression of TNR9 is also linked to the activation of HIV-
1 replication from latently infected CD4+ T cells [50,51].
Upregulation of this receptor in HIV-infected cells may
therefore be essential for the sustained T-cell stimulation
and production of novel proteins that are needed to facil-
itate virus replication and synthesize virus particles with-
out killing the cell. Although the expression of TNF has
been reported in many viral and microbial infections, the
upregulation of this factor in cancer cells has been associ-
ated with the induction of angiogenic factors [52].
Complement Receptor 3 (CO3/C3)
The complement receptor 3 (CO3/C3) was detected only
in HIV-infected cells (Table 1; Figure 2). This protein is the
first responder of the innate immunity and is critical for
the protection of virus-infected hosts/cells. Since amino
acid sequences of human C3 are similar to those of HIV-
gp120 and gp41 envelope proteins, C3 can bind effi-
ciently to different sites on the surface of T-cells and acti-
vate them [53,54]. Expression of C3 in HIV-infected cells
increases the spread of virus to other cell types such as
dendritic cells present in the peripheral blood of HIV-
infected individuals [55-57].
One of the many critical functions of the C3 (and C5
peptidases) is to stimulate chemotaxis and eventually
contribute to the development of choroidal neovasculari-
zation [58,59]. These proteins also enhance permeability
of vasculature and cell migration during embryogenesis (p
= 4 × 10
-4
). Bioinformatics analyses indicates that a coor-

2500
3000
3500
4000
4500
CLR1 LAMA5 VWF
Protein
Mean Value
HIV
Control
Proteins Down-regulated post-HIV- infectionFigure 4
Proteins Down-regulated post-HIV- infection. Two
proteins were downregulated (1433G and PPAC) post-HIV-
infection of T-cells. X-axis = protein abbreviations according
to SwissPROT). Y-axis = average of normalized quantities of
the same protein detected in multiple gels. Error bars repre-
sent one standard deviation for the range of each protein
data. Full protein names and accession #s of each protein are
provided in Table 1.
0
1000
2000
3000
4000
5000
6000
7000
143G PPAC
Protein
Mean Value

T-Cell Activation Pathways Generated by HIV-Modulated ProteinsFigure 5
T-Cell Activation Pathways Generated by HIV-Modulated Proteins. Graphic representation of major proteins and
kinases involved in T-cell activation; the pathways were constructed by the direct Interaction Function Bioinformatics Pro-
grams of Stratagene Pathway Architect 2.0.1. All proteins were uploaded and function-specific pathways were generated auto-
matically; blue outlines around red ovals (ZAP 70, CRKL, and TNR9), indicate the activated proteins. Note numerous cell
surface proteins including PI3K involved in T-cell activation pathways. Lines between red ovals denote major interactions;
green circles represent small molecule interactions. Full names of all protein abbreviations and accession numbers are listed in
Table 1.
Journal of Translational Medicine 2009, 7:75 />Page 10 of 24
(page number not for citation purposes)
pressed) in the HIV-infected T-cells and maintenance of
the overall health and metabolism of activated cells dur-
ing virus replication.
Our bioinformatics analyses indicate that a coordinated
expression of PI3K with protein tyrosine kinases, serine-
threonine kinases and other signaling proteins in our
experimentally HIV-infected cells is critical for the con-
trolled growth of newly made endothelial cells. Thus, con-
comitant expression of cell cycle genes, PI3K, MAPK and
FAK2 together with interacting partners ERBB2, GRB2 and
integrin v-beta (ITB5) in the HIV-infected T-cells is central
to the endothelial cell proliferation which is directly rele-
vant to various biological processes involved in angiogen-
esis. PI3K is also recruited by a phosphotyrosine signaling
complex containing the activated receptor such as ERBB2
and a tyrosine kinase associated adapter protein GRB2
[66]. Another important function of PI3K is its regulatory
role in the formation of tubular structures (vessels) during
angiogenesis [67], through a well-coordinated expression
of ITB5 and cell adhesion molecules that are crucial for

[71]. Activation of this protein controls both the S phase
and G2/M phase checkpoint controls (p = 2.6 × 10
-3
).
Since TP53B also stimulates many different pathways
immediately after the double stranded DNA is perturbed
or damaged [71], it is likely that the integration of HIV
provirus in the cellular DNA may have triggered the
expression of cell-cycle-related pathways through TP53B.
Our bioinformatics and statistical analyses indicate that
activation of TP53B concomitantly with numerous upreg-
ulated transcription factors, growth factors and enzymes
in HIV-infected cells, may be significantly associated with
cell survival and growth (p = 2 × 10
-4
). Further, co-expres-
sion of TP53B with the tyrosine kinase ERBB2, adhesion
molecules, LAMB2 and LAMA5, is also significantly
involved with the formation of vessels during embryonic
development (p = 1.4 × 10
-3
).
Step 3- Augmentation of Cell Growth: Overexpression of Protein
Tyrosine Kinases
The ERBB2 Receptor Protein Tyrosine Kinase
One of the most critical proteins induced by HIV appears
to be the ERBB2 receptor protein tyrosine kinase (ERBB2-
PTK; also known as HER-2/Neu or ERB2) (Table 1; Figure
2). The ERBB2 protein was originally isolated as a viral
oncoprotein, which belongs to the epidermal growth fac-

Journal of Translational Medicine 2009, 7:75 />Page 11 of 24
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development of the embryo. The ERBB2 receptor is acti-
vated by a wide range of pleiotropic growth factors and
induces numerous signal transduction molecules which
stimulate endothelial cell growth during the development
of embryonic organs and angiogenesis [76,77]. A coordi-
nated expression of ERBB2, with GRB2, PI3K, ZAP70 and
FAK-tyrosine kinase and other signaling proteins in the
experimentally HIV-infected cells is therefore anticipated
to activate multiple PTK- regulatory pathways, inhibit
apoptosis, enhance cell survival and stimulate endothelial
cell growth in vivo (p = 3 × 10 – 2 × 10
-7
). These results
indicate that predominant expression of ERBB2-PTK-
activity triggered solely by HIV-replication, without any
other intervention (infection or treatment), represents a
new dimension of VEGF-independent pathways involved
in neovascularization and angiogenesis (p = 4 × 10
-4
). Our
data also suggest that biological processes of angiogenesis
and embryonic development may be driven by common
pathways.
Growth Factor Receptor-Bound Protein 2 (GRB2)
An important cell membrane-associated protein
expressed in HIV-infected cells is the growth factor recep-
tor-bound protein 2 (GRB2) which interacts with the acti-
vated ERBB2 receptor PTK. This protein is essential for the

Suppression of VEGF and its Cognate Receptor Tyrosine Kinase
The VEGF ligand and its cognate receptor VEGFR were not
detected in the experimentally HIV-infected T-cells tested
over a period of two years. Only a single acutely-infected
culture showed basal levels of VEGF-C and its receptor
VEGFR-2 once and was not reproducible in duplicate wells
by MS. The absence in HIV-infected cells was completely
unexpected since the HIV-encoded Tat binds VEGFR via
an arginine-glycine-aspartic-acid (RGD) region of homol-
ogy and activates angiogenic pathways through the PTK
activity of VEGFR [25,81]. However, the RGD domains are
present in numerous integral plasma membrane proteins
identified in this study including integrin and other cell
adhesion proteins [82]. In addition, the binding of Tat to
VEGFR is not as strong as the natural ligand (VEGF) and
the angioproliferative processes are triggered only when
Tat binds VEGFR in the presence of specific factors includ-
ing IL-1 beta, TNF-alpha, IFN-gamma or other angiogenic
cytokines [8,81,83-85].
As discussed above, our data has been corroborated by
unrelated studies in which the expression of ZAP-70-PTK
suppresses VEGF expression [44]. This fundamental knowl-
edge has provided new insights into the tyrosine kinase-
signaling pathways likely to be generated by numerous
PTKs, serine threonine kinases and other signaling pro-
teins identified in the present study. These mechanisms
Protein Tyrosine Kinase and other Major Kinases involved in Angiogenic PathwaysFigure 7
Protein Tyrosine Kinase and other Major Kinases involved in Angiogenic Pathways. Pathways were constructed by
the direct Interaction Function Bioinformatics Programs of Stratagene Pathway Architect. ALL proteins mapped in this figure
have been either upregulated or expressed de novo post-HIV-infection. Proteins were uploaded and function-specific pathways

D pathways and reduces tumor cell proliferation [93].
Downregulation of both VEGF and VEGFR in our HIV-
infected cells could also be attributed to this unique prop-
erty of PKC, as it stabilizes the overexpressed PTK activities
while phosphorylating many proangiogenic protein sub-
strates. Many PKC-beta2 inhibitors are therefore being
tested for a more efficient inhibition of angiogenesis
[94,95].
Our bioinformatics analyses indicate that the presence of
PKC-beta is essential for maintaining an activated state of
major kinases and other signaling proteins (C3, CRKL,
ERBB2, ITGB5, MAPK3, PI3K, and PTK) that are concom-
itantly expressed in HIV-infected cells. This helps the pro-
liferation of endothelial cells while protecting the HIV-
infected cells from apoptosis. In addition, it stabilizes
many critical biological processes necessary for angiogen-
esis (p = 2.6 × 10
-6
).
The Protein Kinase C-binding protein, NELL1
The expression of PKC was accompanied by the upregula-
tion of two of its binding partners NELL1 and Annexin VI
in HIV-infected T-cells (Table 1; Figure 2).
NELL1 is an extracellular matrix glycoprotein which
belongs to a novel class of secreted polymorphic proteins
that control mammalian cell growth and differentiation
in the presence of PKC-beta [96]. The expression of this
versatile protein is important because it contains multiple
EGF-like repeat sequences, thrombospondin (TSP) N ter-
minal sequence and five domains of von Willebrand fac-

[102]. These observations are consistent with our bioin-
formatics findings, indicating that PKC and its binding
partners are vital for regulating the expression of other sig-
naling proteins involved in multiple pathways (p = 2 × 10
-
4
–2 × 10
-7
)
14-3-3 protein gamma (143G)
The amount of PKC expression is regulated by a protein
substrate designated 14-3-3 protein gamma (143G, also
known as PKC inhibitor protein-1) [103,104]. This pro-
tein was downregulated 28% post-HIV infection com-
pared to those present in the uninfected cells (Table 1;
Figure 6). Our results were corroborated by another pro-
teomics-based study in which 143G was downregulated
42-hours post-HIV-infection [29].
143G is an important signaling protein which regulates
cytoskeletal architecture and mediates cellular effects of
protein kinases (especially PKC) by binding specific pep-
tide motifs of proteins that are phosphorylated on serine
or threonine residues [105]. Since PKC is important
kinase for the stability of protein-interactions and contin-
ued T-cell activation, downregulation of 143G may be
essential for regulating and maintaining PKC-related sig-
nals in HIV-infected cells [104,106].
Journal of Translational Medicine 2009, 7:75 />Page 14 of 24
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Step 5- Mitogenic Signaling Cascade: Mitogen-activated Protein

with PI3K-signaling, MAPK regulates angiogenesis and
promotes endothelial cell survival and sprouting [114].
Expression of these kinases is also critical for the cancer
cells as well as for embryonic stem cell growth [86].
The MAPK3 signaling is important for promoting tumor
vascularization in vivo [107]. When MAPK and other fac-
tors are released in the circulation in vivo, they bind to the
cell surface of endothelial cells and activate them. Pro-
longed activation of endothelial cells by MAPK results in
dysregulation of cell adhesion molecules that influence
migration of the newly formed cells via changes in the
cytoskeleton scaffolding [115,116]. These signals also
stimulate smooth muscle proliferation and disrupt cad-
herin-mediated cell-cell interactions, which eventually
promote microvessel formation and vascularization
[101,115,117]. Taken together, our proteomics and bioin-
formatics analyses indicate that a well-synchronized
expression of MAPK3, CRKL, ERBB2, PI3K, PKC, PTK and
numerous adhesion molecules are involved in cell migra-
tion during neovascularization and angiogenesis (p = 3 ×
10
-6
), (Figures 6, 7).
CRK-Like Adapter Protein (CRKL)
The CRK-Like adapter protein (CRKL) is essential for the
activation of MAPK3 and it sustains phosphorylation of
numerous proteins required for mitogenesis, cell prolifer-
ation, differentiation and migration (p = 5 × 10
-5
), [118-

inhibitors -1 and -3 (BAI1 and BAI3 respectively) were
slightly upregulated in HIV-infected cells (Table 1; Figure
2). Both BAI1 and BAI3 are adhesion-type guanine nucle-
otide-binding (G) protein coupled receptors (GPCRs)
essential for mediating receptor tyrosine kinase (PTK) and
GTPase-associated signaling pathways [123,124]. A major
function of these cell-surface receptors is to protect the tis-
sue from increased vascularization by regulating the
expression of excessive proangiogenic factors induced by
various insults such as hypoxia, ischemia, inflammation
or tumorigenesis (p = 7 × 10
-7
), [125-127].
Journal of Translational Medicine 2009, 7:75 />Page 15 of 24
(page number not for citation purposes)
The roles of BAI1 and BAI3 in HIV-infected human cells
are not clear. However, in the human brain, BAI1 is a p53-
target gene important for signal transduction [128,129].
Our bioinformatics analyses suggest that these GPCRs
may be similar to other "embryonic" proteins that have
been dysregulated by HIV-infection and may be necessary
to sustain different PTK-mediated cellular processes
involved in cell-adhesion and protein-protein interac-
tions necessary for enhanced virus replication, cell
growth, migration and invasion. Expression of BAI1 and
BAI3 receptors in HIV-infected T-cells also suggests that
both proangiogenic and anti-angiogenic signals are neces-
sary for maintaining a balance of tyrosine kinase phos-
phorylation and focal adhesion signaling to restrict
pathologic angiogenesis [125,126,129]. The BAI1 protein

sized endothelial cells to adhere together in vivo as they
tend to differentiate into functional entities [2,91]. Thus,
FAK2 plays a vital role in endothelial cell growth, prolifer-
ation, survival, motility, migration and differentiation (p
= 2 × 10
-4
), [119,137,138].
Expression of adhesion molecules is also essential for ang-
iogenesis in the embryo (p = 4 × 10 – 2 × 10
-7
).
The numerous diffusible factors described in this study
provide compelling evidence that binding of several
members of adhesion molecules to their cognate receptors
on the endothelial cells in vivo would be expected to pro-
mote FAK2 tyrosine kinase-coordinated signals for
endothelial cell proliferation, adhesion, morphogenesis
and angiogenesis [119,120,134]. Our bioinformatics and
statistical analysis indicates that the FAK2- PTK activity
alone is critical for angiogenic processes (p = 2.6 × 10
-3
).
A well-coordinated expression FAK2 with other protein
tyrosine kinases (ZAP70, ERBB2, ITB5), and many
adapter/signaling proteins in HIV-infected cells is highly
significant for angiogenesis (p = 1.3 × 10
-5
).
Integrin alpha-v- beta-5 (ITB5) and Fibronectin (FINC)
Both integrin alpha-v-beta-5 (ITB5) and fibronectin

-8
).
The integrin was synchronously upregulated in HIV-
infected cells with numerous cell-surface signaling pro-
Journal of Translational Medicine 2009, 7:75 />Page 16 of 24
(page number not for citation purposes)
teins such as ERBB2, PI3K discussed earlier. These findings
are in agreement with the report that PI3K signaling path-
ways are initiated by ERBB which upregulates beta1-
integrin functions [146]. Thus, the overexpression of
ERBB-PTK, GRB2, ZAP-70, MAPK, dysregulation of
integrins and upregulation of adhesion kinase, all contrib-
ute to the formation of vasculature and promote angio-
genesis via novel VEGF-independent pathways (Table 1)
[82,139].
Expression of Nitric-oxide Synthase (NOS) and Downregulation of
PPAC
A critical enzyme expressed in our experimentally infected
cells was the nitric oxide synthase (NOS or NS2A) (Figure
2). This enzyme is located in the plasma membrane and
transported to the cytoplasm to regulate multiple func-
tions [147]. NOS is activated in response to cellular stress
and it regulates vascular functions including endothelial
cell migration necessary for angiogenesis [147].
Expression of NOS in HIV-infected cells is considered to
be important as it also inactivates the low molecular
weight phosphotyrosine protein phosphatase (PPAC, Syn.
HCPTPA), an enzyme that impairs the VEGF-mediated
autophosphorylation [36,148]. Although PPAC phosphatase
was detected in the uninfected T-cells, its expression was

laminin alpha-5 chain (LAMA5) was expressed in both
the HIV-infected and uninfected control cells, and only
LAMB2 was upregulated in HIV-infected cells. Laminin
beta-3 chain (LAMB3 precursor) and laminin gamma-1
chain (LAMC1) were detected only once at low levels and
therefore were not included in the analyses.
Laminins are a family of morphogenic glycoproteins,
which are secreted and incorporated into the extracellular
matrices of many tissues. These proteins bind to different
isoforms of integrins and other cell surface receptors to
form cellular structural scaffoldings [149,150].
Thus, LAMB2, which is present in the basement mem-
branes of many tissues, is essential for cell proliferation,
migration and differentiation of cells in early develop-
ment of embryos [149]. This protein has EGF-like extra-
cellular domains crucial for rolling up and adhesion of
endothelial cells to form microvessels [151]. Statistical
analysis shows that the coexpression of LAMB2, MAPK3,
CRKL, FAK2, with ERBB2, GRB2, INC, NOS2 TNR9,
MYLK, PKC, TP53BP1 and numerous PTK signaling pro-
teins is highly significant for the survival, morphogenesis,
migration and microvessel formation of cells (p = 6 × 10
-
7
) [25,131,152-154].
Cadherin EGF LAG Seven-Pass G-Type Receptor 1 (CLR1/CELSR1)
Among the membrane-bound proteins that were upregu-
lated in HIV infected T-cells, cadherin EGF LAG seven-pass
G- coupled protein receptor (GPCR) type 1
(CELSR1,syn.CLR1) was detected frequently in HIV-

Protocadherin Focal Adhesion Targeting type 2 (FAT2) Protein
The protocadherin focal adhesion targeting (FAT) protein
type 2 belongs to a novel superfamily of membrane asso-
ciated cadherins. FAT2 was expressed exclusively in HIV-
infected cells (Table 1) and is homologous to Drosophila
FAT proteins (FAT1, FAT2, FAT3 and FAT4) [158,159].
Expression of FAT2 is essential for cell recognition, regula-
tion of polarity during cell adhesion, microvessel forma-
tion and correct morphogenesis of the embryo [159,160].
Protocadherins also regulate angiogenesis in specific
brain regions or a subset of blood vessels in the develop-
ing vertebrate brain [157,158]. However, expression of
FAT2 mRNA in adults is associated with numerous can-
cers such as highly metastatic/angiogenic ovarian and
head and neck cancers [158].
Golgi apparatus Protein 1 (GLG1)
The Golgi apparatus protein 1 (GLG1) was expressed
exclusively in HIV-infected cells (Table 1; Figure 2). GLGI,
also known as E-selectin-type integral membrane protein,
Golgi sialoglycoprotein (MG-160), E-selectin ligand 1
(ESL-1) or cysteine-rich fibroblast growth factor (FGF)
receptor CFR-1, is normally expressed on endothelial cells
and mediates morphogenesis and trafficking of cells
through the vascular endothelium (p = 2 × 10
-5
), [161].
The expression of GLG1 is enhanced on lymphocytes that
are in contact with the endothelium, because it interacts
with adhesion molecules and their cognate receptors
present on the endothelial cell [162].

ThromboSpondin (TSP)-Type I sequence motifs), contain
an ADAM protease domain [165] as well as throm-
bospondin 1 repeats [166,167]. This protein was
expressed in HIV-infected T-lymphocytes (Table 1).
Morphogenesis of cellular structures requires well-con-
trolled proteolytic activities that are regulated by protein-
ases. ADAMTS are specific metalloproteases or
aggrecanase localized in the extracellular space critical of
the cleavage of large aggregating proteoglycans or aggre-
cans normally expressed in growing tissues [167,168].
Compared to other aggrecanase, ADAMTS-9 is more
responsive to proinflammatory cytokines, such as TNF
and chemokines expressed in HIV infected cells in vitro or
in vivo [169].
An altered expression of ADAMTS enzyme contributes to
the permeability and migration of cells from tissues, a fea-
ture essential for microvessel formation [167,170].
ADAMTS- 9 can punctuate basement membranes of the
endothelial cells in front of the sprouting vessel such that
the proliferating cells can penetrate existing vessels
through the small microscopic perforations [166].
The TSP-containing proteins were initially reported to
exhibit anti-angiogenic and tumor suppressor activities in
mice [171], ADAMTS- matrix metalloproteinases with
thrombospondin repeats have since been considered
important factors for angiogenesis and other endothelial
cell functions [172]. Thus, co-expression of ADAMTS9,
C3, FN1, MAPK3, PKC, TNFR9 and TP53BP1 in the pres-
ence of ERBB2, LAMB2 and other proteins in the experi-
mentally infected cells is significantly associated with

).
Step 10- Preservation of Differentiated Endothelial Cells: Von
Willebrand Factor
Von Willebrand Factor (VWF)
The Von Willebrand factor (VWF) binds to platelet recep-
tors and activates these cells [175]. The VWF- precursor
was upregulated in the experimentally HIV-infected T-
cells, compared to the uninfected counterpart cells (Table
1). This factor is normally produced by endothelial cells
and secreted in the plasma. Diverse physiological func-
tions performed by VWF include cell adhesion, cell migra-
tion, cell cycle progression and differentiation of
endothelial cells [175-178]. The VWF also acts as a perme-
ability barrier for endothelial cells and is vital for the
transport of the coagulation factor VIII in the plasma
[178].
While an increased expression of VWF has been linked
directly or indirectly to HIV infection of endothelial cells
[179], it also augments activation and adhesion of aggre-
gated platelets and interacts with integrins and FINC in
order to maintain cellular integrity (Figure 8) [180].
Enhanced production of VWF is also indicative of vascular
injury, thrombus formation, inflammation and angiogen-
esis [176,177]. In HIV-infected individuals an increase in
the plasma levels of VWF is considered a marker of
endothelial cell proliferation resulting in abnormal pat-
terns of angiogenesis [181]. Patients with highly dysplas-
tic anal warts, cervical and vulvar cancers also show
statistically significant correlations with the upregulation
of VWF and enhanced capillary formation, microvessel

2. Based on the protein-protein interaction pathway anal-
yses, we have identified key events during angiogenesis
and proposed comprehensive putative mechanisms by
which a well-coordinated expression of several families of
proteins (cell surface receptors, kinases, regulatory
enzymes, growth factors, adhesion molecules and other
signaling proteins) can generate a network of interactions
along multiple novel pathways leading to T-cell activa-
tion, transcriptional and translational reprogramming,
cell cycle changes, cell proliferation, cell growth, migra-
tion, cell adhesion, sprouting, microvessel formation and
maintenance of differentiated endothelial cells that are
highly significant for neovascularization or angiogenic
responses (p = 1.0 × 10
-11
).
3. While the in vitro results cannot be correlated directly to
the consequences of HIV-infection in vivo, a unique find-
ing of our bioinformatics analyses is that activation of T-
cells results in the production of a diverse array of protein
tyrosine kinases (PTKs), serine-threonine kinases, lipid
kinases, adhesion molecules and other diffusible signal-
ing proteins. The abundance of multiple PTKs and other
kinases initiates novel angiogenic pathways independent of
VEGF-signaling while suppressing activation of VEGFR-PTK
activity. This mechanism is similar to that observed in
neovascularization in the developing embryo.
4. Since T-cells and monocytes/macrophages are the pri-
mary cell types to be infected at the portal of entry in vivo,
the HIV-infected T-cells may induce ERBB2 and other

Author's information
Suraiya Rasheed is a Professor of Pathology and Director,
Laboratory of Viral Oncology and Proteomics Research at
the Keck School of Medicine, University of Southern Cal-
ifornia, Los Angeles. She has expertise in molecular biol-
ogy of HIV and proteomics research. Her laboratory
discovered the first ras oncogene in the form of the Rash-
eed Rat Sarcoma virus and the Feline Gardner Rasheed
(Fgr) oncogene in a feline sarcoma virus. This laboratory
has also isolated a novel HIV strain (HIV-Ibng) from
Nigeria, a unique cat endogenous retrovirus (RD114) and
the naturally occurring amphotropic murine leukemia
Proteins Involved in Preservation of Differentiated Endothelial Cell PhenotypesFigure 8
Proteins Involved in Preservation of Differentiated Endothelial Cell Phenotypes. Protein-interaction pathways
responsible for maintaining differentiated state of endothelial cells. Full names of all protein abbreviations and accession num-
bers are listed in Table 1.
Journal of Translational Medicine 2009, 7:75 />Page 20 of 24
(page number not for citation purposes)
viruses that replicate in human cells. These retroviruses
are used globally for constructing vectors for gene transfer.
Bruce Lai is a computer scientist and is an expert in mass
spectrometry; Jasper Yan and Adil Hussain are students.
Acknowledgements
We acknowledge technical help of Zisu Mao and Jane M.C. Chan for the
performance of two-dimensional gel electrophoresis and mass spectrome-
try respectively, we thank the technical-support personnel of IPA-Ingenuity
Systems (Bioinformatics Programs) are acknowledged for answering ques-
tions and manufacturers of Strategene Pathway Architect program for ena-
bling us to use their program for a short time. Thanks are also due to Karen
Lau for her help in constructing some protein-interaction pathways, Rahim

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